Energy balance (involving energy metabolism, nutritional state, lipid storage and the like) is an important criteria for health. This energy homeostasis involves food intake and metabolism of carbohydrates and lipids to generate energy necessary for voluntary and involuntary functions. Metabolism of proteins can lead to energy generation, but preferably leads to muscle formation or repair. Among other consequences, a lack of energy homeostasis lead to over or under formation of adipose tissue.
Formation and storage of fat is insulin-modulated. For example, insulin stimulates the transport of glucose into cells, where it is metabolized into xcex1-glycerophosphate which is used in the esterification of fatty acids to permit storage thereof as triglycerides. In addition, adipocytes (fat cells) express a specific transport protein that enhances the transfer of free fatty acids into adipocytes.
Adipocytes also secrete several proteins believed to modulate homeostatic control of glucose and lipid metabolism. These additional adipocyte-secreted proteins include adipsin, complement factors C3 and B, tumor necrosis factor xcex1, the ob gene product and Acrp30. Evidence also exists suggesting the existence of an insulin-regulated secretory pathway in adipocytes. Scherer et al., J. Biol. Chem. 270(45): 26746-9, 1995. Over or under secretion of these moieties, impacted in part by over or under formation of adipose tissue, can lead to pathological conditions associated directly or indirectly with obesity or anorexia.
Acrp30 is a 247 amino acid polypeptide that is expressed exclusively by adipocytes. The Acrp30 polypeptide is composed of a amino-terminal signal sequence, a 27 amino acid stretch of no known homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen repeats and a carboxy terminal globular domain. See, Scherer et al. as described above and International Patent Application No. WO 96/39429. Acrp30, an abundant human serum protein regulated by insulin, shares structural similarity, particuliarly in the carboxy-terminal globular domain, to complement factor C1q and to a summer serum protein of hibernating Siberian chipmunks (Hib27). Expression of Acrp30 is induced over 100-fold during adipocyte differentiation. Acrp30 is suggested for use in modulating energy balance and in identifying adipocytes in test samples.
Another secreted protein that appears to be exclusively produced in adipocytes is apM1, described, for example, in Maeda et al., Biochem. Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone had a 244 amino acid open reading frame and a long 3xe2x80x2 untranslated region. The protein included a signal sequence, an amino-terminal non-collagenous sequence, 22 collagen repeats (Gly-XAA-Pro or Gly-Xaa-Xaa), and a carboxy-terminal region with homology to collagen X, collagen VIII and complement protein C1q.
Complement factor C1q consists of six copies of three related polypeptides (A, B and C chains), with each polypeptide being about 225 amino acids long with a near amino-terminal collagen domain and a carboxy-terminal globular region. Six triple helical regions are formed by the collagen domains of the six A, six B and six C chains, forming a central region and six stalks. A globular head portion is formed by association of the globular carboxy terminal domain of an A, a B and a C chain. C1q is therefore composed of six globular heads linked via six collagen-like stalks to a central fibril region. Sellar et al., Biochem. J. 274: 481-90, 1991. This configuration is often referred to as a bouquet of flowers. Acrp30 has a similar bouquet structure formed from a single type of polypeptide chain.
Molecules capable of modulating energy homeostasis are sought for the study of this phenomena and for the prevention or treatment of imbalances. Also, molecules capable of modulating adipocyte secretory pathways are also sought as indirect energy homeostasis modulators and as research reagents.
The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect the invention provides an isolated polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion. Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2. Within a related aspect, the polypeptide is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO:2. Within another embodiment the polypeptide comprises residues 1-281 of SEQ ID NO:2 or SEQ ID NO:44. Within another embodiment the polypeptide is covalently linked amino terminally or carboxy terminally to a moiety selected from the group consisting of affinity tags, toxins, radionucleotides, enzymes and fluorophores. Within a related embodiment is further provided a proteolytic cleavage site between said sequence of amino acid residues and said affinity tag.
Within another aspect is provided an isolated polypeptide selected from the group consisting of: a) a polypeptide having a sequence of amino acid residues that is 75% identical in amino acid sequence to amino acid residue 99 to amino acid residue 140 of SEQ ID NO:2; b) a polypeptide having a sequence of amino acid residues that is 75% identical in amino acid sequence to amino acid residue 140 or 141 to amino acid residue 281 of SEQ ID NO:2; and c) a polypeptide having a sequence of amino acid residues that is 75% identical in amino acid sequence to amino acid residue 99 to 281 of SEQ ID NO:2.
Within another aspect is provided a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion comprising a polypeptide selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 26 to amino acid residue 281 of SEQ ID NO:2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1, 22 or 26 to amino acid residue 281; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:44 from amino acid residue 1, 22 or 26 to amino acid residue 281; d) a portion of the zsig37 polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:44, containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig37 polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:44 containing the globular-like domain or an active portion of the globular-like domain; or e) a portion of the zsig37 polypeptide as shown in SEQ ID NQ:2 or SEQ ID NO:44 including the collagen-like domain and the globular domain; and said second portion comprising another polypeptide. Within one embodiment the first portion is selected from the group consisting of: a) a polypeptide having the sequence of amino acid residue 99 to amino acid residue 140 of SEQ ID NO: 2 or SEQ ID NO:44; b) a polypeptide having the sequence of amino acid residue 140 or 141 to amino acid residue 281 of SEQ ID NO: 2 or SEQ ID NO:44; c) a polypeptide having the sequence of amino acid residue 99 to 281 of SEQ ID NO:2 or SEQ ID NO:44.
Within another aspect is provided a fusion protein. comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-21 or 1-25 of SEQ ID NO:2 or SEQ ID NO:44, wherein said secretory signal sequence is operably linked to an additional polypeptide.
Within a further aspect is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a transcription terminator. Within one embodiment the DNA segment encodes a polypeptide that is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2. Within another embodiment the DNA segment encodes a polypeptide that is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO:2. Within another embodiment the DNA segment encodes a polypeptide comprising residues 1-281 of SEQ ID NO:2 or SEQ ID NO:44. Within yet another embodiment the DNA segment encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag. Within a further embodiment the DNA segment further encodes a secretory signal sequence operably linked to said polypeptide. Within yet another embodiment the secretory signal sequence comprises residues 1-21 or 1-25 of SEQ ID NO:2 or SEQ ID NO:44.
Within another aspect is provided a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a transcription terminator, wherein said cell expresses said polypeptide encoded by said DNA segment.
Within still another aspect is provided a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion; and a transcription terminator; whereby said cell expresses said polypeptide encoded by said DNA segment; and recovering said expressed polypeptide.
Within another aspect is provided a pharmaceutical composition comprising a polypeptide, said polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion; in combination with a pharmaceutically acceptable vehicle.
Within still another aspect is provided an antibody that specifically binds to an epitope of a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion.
Within another aspect is provided a binding protein that specifically binds to an epitope of a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion.
Within another aspect is provided an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2, wherein said sequence comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid; and a carboxy-terminal globular portion. Within one embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO:2. Within another embodiment the polypeptide is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO:2. Within another embodiment the polypeptide comprises residues 1-281 of SEQ ID NO:2 or SEQ ID NO:44. Within another embodiment the polynucleotide is DNA.
Within another aspect is provided an isolated polynucleotide selected from the group consisting of, a) a sequence of nucleotides from nucleotide 465 to nucleotide 688 of SEQ ID NO:1; b) a sequence of nucleotides from nucleotide 688 to nucleotide 1016 of SEQ ID NO:1; c) a sequence of nucleotides from nucleotide 691 to nucleotide 1016 of SEQ ID NO:1; d) a sequence of nucleotides from nucleotide 465 to nucleotide 1016 of SEQ ID NO:1; e) a sequence of nucleotides from nucleotide 364 to nucleotide 490 of SEQ ID NO:43; f) a sequence of nucleotides from nucleotide 490 to nucleotide 912 of SEQ ID NO:43; g) a sequence of nucleotides from nucleotide 364 to nucleotide 912 of SEQ ID NO:43; h) a sequence of nucleotides from nucleotide 364 to nucleotide 490 of SEQ ID NO:43; i) a polynucleotide encoding a polypeptide having a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 99, 140 or 141 to amino acid residue 281 of SEQ ID NO:2; j) a polynucleotide encoding a polypeptide having a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 99 to amino acid residue 140 of SEQ ID NO:2; k) nucleotide sequences complementary to a), b), c), d), e), f), g), h), i) or j) and l) degenerate nucleotide sequences of a), b), c), d), e), f), g), h), i), j) or k).
Within another aspect is provided an isolated polynucleotide encoding a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion is selected from the group consisting of: a) a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to amino acid residue 26 to amino acid residue 281 of SEQ ID NO:2; b) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:2 from amino acid residue 1, 22 or 26 to amino acid residue 281; c) a polypeptide comprising a sequence of amino acid residues as shown in SEQ ID NO:44 from amino acid residue 1, 22 or 26 to amino acid residue 281; d) a portion of the zsig37 polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:44, containing the collagen-like domain or a portion of the collagen-like domain capable of dimerization or oligomerization; e) a portion of the zsig37 polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:44 containing the globular-like domain or an active portion of the globular-like domain; or f) a portion of the zsig37 polypeptide as shown in SEQ ID NO:2 or SEQ ID NO:44 including the collagen-like domain and the globular domain; and said second portion comprising another polypeptide.
Within yet another aspect is provided an isolated polypeptide encoding a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues. 1-21 or 1-25 of SEQ ID NO:2 or SEQ ID NO:44, wherein said secretory signal sequence is operably linked to an additional polypeptide.
Within yet another aspect is provided an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:23 or a sequence complementary to SEQ ID NO:23.